Many of the signaling cascades of eukaryotes are perpetuated throughout the cell via non-catalytic protein-protein interactions. These interactions are made possible by the presence of conserved complementary domains or modules within the signaling proteins. Adapter proteins, characterized by the presence of one or more such binding domains, function by mediating the rapid and specific assembly of multi-protein complexes during signal transduction. Several conserved protein domains have been identified, the best characterized being the src homology domains (SH2 and SH3). SH2 and SH3 complexes couple tyrosine phosphorylation events, via interaction with proline-rich motifs, to downstream effectors (Pawson, Nature, 1995, 373, 573-580).
Nck-2 (also known as Nck adapter protein, Nck-beta, hNck-beta and Grb4) is the most recent member of the Nck family of adapter proteins to be identified. Like the other member of the family, nck-1, nck-2 also contains both SH2 and SH3 domains and associates with proteins involved in integrin and receptor tyrosine kinase signaling, in particular growth factor receptor pathways (Braverman and Quilliam, J. Biol. Chem., 1999, 274, 5542-5549; Chen et al., J. Biol. Chem., 1998, 273, 25171-25178; Tu et al., Mol. Cell. Biol., 1999, 19, 2425-2434; Tu et al., Mol. Biol. Cell, 1998, 9, 3367-3382). While Nck proteins have been shown to be expressed in all tissues examined, they do show differential expression patterns. Nck-2 shows higher expression in heart, brain, placenta, skeletal muscle and pancreas (Braverman and Quilliam, J. Biol. Chem., 1999, 274, 5542-5549; Chen et al., J. Biol. Chem., 1998, 273, 25171-25178; Tu et al., Mol. Biol. Cell, 1998, 9, 3367-3382). Nck-2 interacts with both the epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors and inhibits EGF and PDGF-stimulated DNA synthesis in an SH2-dependent manner (Chen et al., J. Biol. Chem., 1998, 273, 25171-25178).
The primary contacts made between cells and the surrounding environment, including contacts with other cells, are mediated by the transmembrane proteins known as integrins. These contacts are critical for the bidirectional transduction of signals to the interior of the cell and consequently to the modulation of biochemical pathways. Tu et al. have demonstrated that nck-2 participates in pathways that connect growth factor receptor-signaling and integrin signaling (Tu et al., Mol. Biol. Cell, 1998, 9, 3367-3382). In these studies, it was shown that nck-2 interacts with PINCH, an adapter protein containing cystein-rich motifs known as LIM domains, which is involved in integrin, growth factor and Wnt signaling pathways (Tu et al., Mol. Cell. Biol., 1999, 19, 2425-2434).
In mice, nck-2 has been implicated in the development of cancer by interacting with the protein products of the Ras and BCR/Abl genes, genes known to have activating mutations in human malignancies such as leukemia and breast cancer. Bravermann et al. have shown that nck-2 cooperates with v-Abl and Sos to induce transcriptional activation as well as with Ras and v-Abl to induce transformation of NIH3T3 cells (Braverman and Quilliam, J. Biol. Chem., 1999, 274, 5542-5549). The pharmacological modulation of nck-2 expression and/or function may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of nck-2 and to date, investigative strategies aimed at modulating nck-2 function have involved the use of antibodies and molecules that block protein-protein interactions. Disclosed in U.S. Pat. No. 5,780,496 are methods and compositions intended for use in the inhibition of adapter protein-tyrosine kinase interactions. These compounds, quinazoline derivatives, are designed to interfere with molecular interactions that occur through SH2 or SH3 domains to ameliorate cell proliferative disorders (Tang et al., 1998). In addition, methods for assaying for a substance that affects an SH2-regulated signaling system are disclosed in U.S. Pat. No. 5,352,660 (Pawson, 1994). These methods comprise providing a Src homology region of any of several SH2 containing proteins along with an SH2-phosphorylated ligand such that the complex formed activates an SH2-phosphorylated ligand regulatory system. However, there remains a long felt need for additional agents capable of effectively inhibiting nck-2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of nck-2 expression.
The present invention provides compositions and methods for modulating nck-2 expression.